A hapten may be defined as a chemical composition of limited molecular weight (usually less than 1000) which in and of itself does not elicit antibody formation when introduced into a host animal. However, when covalently bonded to a high molecular weight antigenic carrier, the resultant hapten-carrier conjugate can elicit in the host animal the formation of antibodies which recognize the hapten composition. Examples of haptens to which antibodies have been raised in this fashion are numerous, including such classes of materials as medicaments (therapeutic drugs), amino acids and metabolites, small peptides, steroids and aromatic residues such as the dinitrophenyl moiety. Typical carriers are large polyvalent molecules such as proteins, polysaccharides and glycoproteins which are not native to the host animal. The methods for preparation of hapten-carrier conjugates are well known in the art and have been reviewed (B. F. Erlanger; Methods in Enzymology, v. 70 , p. 84 , 1980).
A divalent hapten may be considered as a subclass of hapten-carrier conjugates. The carrier, which may be of any molecular weight, is covalently bonded to the haptens by means of only two functional groups. The two functions may be different but they are generally the same. The carrier molecule also may be of any molecular weight or of any molecular shape.
The carriers disclosed herein function primarily to separate the two hapten moieties by a chemical spacer. Because of their divalent character they can bridge antigen binding sites on adjacent antibody molecules directed towards hapten, thus differentiating them from free hapten.
The concept of divalent haptens for bridging adjacent antibody molecules was investigated by R. C. Valentine and N. M. Green for a series of 2,4-dinitrophenylated polymethylenediamines (J. Molecular Biol., 27, 615 1967). It was found that a minimum of an eight carbon length spacer allowed bridging of antibodies. However, the water solubility of the spacers described by Valentine and Green was found to be very poor. A divalent hapten system which is more water soluble is needed and desired.
In recent times, particular interest has been directed at a clinical assay method of diagnosis in which the pathological state or the pathological prognosis of a patient is assessed, or the medicament dose which is to be administered to the patient is determined, in such a manner that the content in the urine or blood of a physiologically active substance which has a low molecular weight and is in this body in the form of a hapten (and its metabolites), or of a medicament (and its metabolites) which has been administered to the patient, is measured.
Examples of the haptens to be measured in carrying out this clinical assay method or diagnosis include thyroid hormones such as, for example, L-3,3'5-tri-iodothyronine (T.sub.3) and L-thyroxine (T.sub.4).
The medicaments which are to be measured in this assay method or diagnosis include medicaments whose dose should be determined with extreme care and whose actions exhibit a relation to their concentration in the blood or urine. Some typical medicaments include digitalis preparations, antibiotics such as tetracycline, psychotropic agents such as amphetamine, narcotics such as morphine, blood coagulants and anticoagulants, and in particular theophylline and phenobarbital.
The haptens are usually found in trace quantities, being present in blood or urine as complex-bound or conjugated forms with complicated compositions. For this reason, an elaborate and time-consuming method to determine and measure them is required.
A variety of methods can be used to measure these haptens, namely physicochemical methods, immunochemical methods or competitive protein-binding methods.
Immunochemical methods are now considered superior to physicochemical methods due to the reaction specificity and sensitivity of measurement. A large number of immunochemical methods for the measurement of traces of haptens in the human body are known at present, for example agglutination inhibition methods, radioimmunoassay (RIA) and enzyme immunoassay (EIA) . In a known EIA method, use is made of particles which are loaded with the antibody to the hapten which is to be measured and of a coupling product of the hapten with a carrier substance (See, German Offenlegungsschrift 2,155,658 and German Patent 2,743,445).
The procedure for an agglutination inhibition method entails the use of a coupling product of a hapten, which is the same hapten as the hapten which is to be measured, and a carrier substance, such as a protein, a polysaccharide or a glycoprotein, bound to solid particles, for example blood cells or latex with a high molecular weight, as the antigen. An antibody to the hapten which is to be measured is obtained from an antiserum which is obtained from mammals, for example guinea pigs, rabbits or sheep, which have been immunized with the coupling product of the hapten and the carrier substance.
When the antibody is mixed with the fine particles onto which the hapten-carrier conjugates are coupled an agglutination reaction between these two components takes place. The agglutination reaction is inhibited when the hapten which is to be measured is present in the sample. This method is an extremely straightforward method which is able to measure the hapten which is present in the blood or urine in the form of a complex or conjugate, without the necessity for complicated methodological measures, Such as hydrolysis or chromatography. The sensitivity of this method is about 100 ng/ml, even if blood cells are used as the particles. However, since most haptens which are of interest to measure in the human body are present in quantities between 500 pg/ml and 50 ng/ml , they must be concentrated in order to carry out successful measurement.
It is desired to develop a new immunochemical method for the measurement of haptens by inhibition of agglutination which exhibits higher sensitivity of measurement than the heretofore known methods.